THIS INVENTION relates to an assay method and apparatus suitable for determination of analyte wherein assay apparatus is provided having one or multiple capture zones incorporating a receptor ligand for detection of analyte.
Lateral flow membranes for immunoassays are well known and reference may be made, for example, to U.S. Pat. No. 4,943,522 which refers to a porous membrane capable of non-bibulous lateral flow which is used as an assay substrate. A member of a binding pair is affixed in an indicator zone defined in the substrate. A sample is applied at a position distant from the indicator zone and permitted to flow laterally through the zone. Any analyte in the sample is complexed by the affixed binding partner and detected. A method of detection employs entrapment of observable particles in the complex.
Another lateral flow assay is described in U.S. Pat. No. 5,798,273 wherein-the presence or amount of an analyte in a sample may be determined by contacting a sample with anti-analyte antibodies which bind to the analyte or to both analyte and an analyte analog and an assay tracer prior to application to a lateral flow device. A first member of a binding pair is incorporated with the tracer. The lateral flow device comprises a solid support having a sample addition area, a capture area containing analyte or analyte analog which is immobilized thereon and a read out area comprising one or a plurality of zones having a second member for the binding pair immobilized thereon which binds with the first member to detect the presence or amount of analyte in the sample.
Lateral flow type assays are also described in European Patent No. EP0291194 which refers to a casing having a dry porous carrier. A liquid test sample may be applied to the dry porous carrier which has a labelled specific binding reagent for an analyte which reagent is freely movable within the porous carrier when in the moist state and unlabelled specific binding reagent for the same analyte which is immobilized in a detection zone on the porous carrier. The liquid sample may pick up labelled reagent and thereafter permeate to the detection zone. The label used is a particulate direct label.
Reference may also be made to lateral flow assay devices which are described in U.S. Pat. Nos. 5,607,863, 5,648,274 and 5,468,648 which each refer to devices having a first opposable component and a second opposable component wherein at least one of the opposable components comprises capture and detection zones and at least one of the opposable components includes an absorber for contacting the other of the opposable components for detection of analyte. Each of these references describe capture zones which incorporate specific binding partners for the analyte. U.S. Pat. No. 5,468,648 describes provision of a test strip that can carry out multiple assays simultaneously.
Reference may also be made to U.S. Pat. Nos. 5,401,667, 4,855,240, 5,602,037, 5,712,170 and 4,727,019 which all refer to complicated forms of lateral flow assay devices and each of which incorporates capture zones for capture reagent or receptor to bind directly to analyte in a sample being tested.
Reference may also be made to U.S. Pat. No. 5,185,127 which refers to a vertical filter stack in an enclosure having a base portion and a lid. The filter stack has an initial hydrophilic membrane having a binder for analyte thereon, a hydrophobic membrane under the hydrophilic membrane and a pad of absorbent material under the hydrophobic membrane.
Reference may also be made to the following references which use a lateral flow device described above in U.S. Pat. No. 5,468,648, i.e.:
(i) Chew et al., 1998, Clin. Diagn. Lab. Immunol. 5 407-409;
(ii) Vaughn et al., 1998, J. Clin. Micro. 36 234-238;
(iii) Lam et al., 1998, J. Clin. Virol. 10 75-81;
(iv) Cuzzubbo et al.., Jan/Mar 1998, Singapore Microbiologist, 16-17; and
(v) Devine et al., 1998, Analytical Testing Technology 15 16.
As is clear from the prior art referred to above, a feature of such prior art is the provision of lateral flow devices or filter stacks which include one or a plurality of capture zones for direct binding of analyte for detection of the analyte.
However, it has now been realised that such prior art devices have several disadvantages, the most notable of which is the following:
(i) by employing capture zones having a specific binding agent for the analyte, it will be appreciated that such binding agents, which are usually proteins, may in some cases comprise unstable biological material that is usually incapable of being stored at room temperature;
(ii) by virtue of (i), it is usual that such prior art devices can only be used once and therefore must be discarded;
(iii) only relatively small volumes of sample and accompanying reagents can be accommodated due to the necessity for controlling the flow of sample and accompanying reagents through the prior art device to relatively slow speeds;
(iv) often accumulation of complexes occur both in the capture zones and non-capture zones restricting the flow of sample and reagents through the prior art device;
(v) often non-specific reactions and aggregation of reactants may cause non-specific binding; and
(vi) serious limitations apply to processing detection of multiple analytes in prior art devices. For example, when detection of both multiple specificities of IgG and IgM classes of antibodies is being attempted, it is not possible to use anti-IgG or anti-IgM receptors in the capture zones or antigens specific to IgG or IgM antibodies because of the danger of cross reactions occurring, preventing the differentiation between antibody specificity and immunoglobulin class.
It is therefore an object of the invention to provide an assay method to reduce the affect of the abovementioned disadvantages.
It is a further object of the invention to provide an assay apparatus which is of simple construction and which can be used for assaying a number of different analytes simultaneously.
In accordance with the invention, there is provided a method of assaying for an analyte including the steps of:
(i) mixing a liquid sample suspected of containing an analyte and reagents comprising a target ligand analyte receptor conjugate and a detectable tracer containing a label for the analyte whereby if the analyte is present, there is formed in solution a complex of analyte coupled to the conjugate after incubation of the mixture;
(ii) passing the mixture through filter apparatus containing a plurality of discrete flow zones wherein at least one zone functions as a capture zone having bonded thereto a receptor ligand for the target ligand;
(iii) detecting the presence of analyte in the sample by activation of the label in said at least one zone after binding of the complex to the receptor ligand; and
(iv) continuing the passage of the mixture past said at least one zone to a wicking zone for liquid entrapment.
Thus, one of the principal features of the present invention is that any analyte complex(es) involving the target ligand-analyte receptor conjugate are formed in a liquid or fluid phase prior to retention on a capture zone when the complex(es) bind to associated receptor ligand(s).
The above method is especially adapted for assaying a number of different analytes simultaneously. To this end, the assay apparatus may have a plurality of capture zones which have a different receptor ligand bonded thereto. The sample in step (i) may also be combined with a target ligand-analyte receptor conjugate specific for each analyte being assayed and a detectable tracer incorporating a label for each analyte. In this embodiment, step (ii) facilitates the activation of each analyte by detection of the label in each zone after bonding of each complex to its associated receptor ligand.
The method of the invention may be used for detection of a wide variety of different analytes. In this regard, it will be appreciated that the term xe2x80x9canalytexe2x80x9d refers to any compound or composition to be detected that is capable of binding specifically to a binding agent. Examples of binding agents are antibodies, antigens or chelating agents or receptor ligands discussed hereinafter.
The term xe2x80x9canalytexe2x80x9d may also include within its scope analyte analogs as referred to in U.S. Pat. No. 5,624,809 which is herein incorporated by reference in its entirety.
Analytes may have a simple binding site (i.e. monovalent) or multiple binding sites (i.e. polyvalent). Polyvalent analytes may comprise polymers inclusive of polypeptides, proteins, polysaccharides, nucleic acids or combinations thereof.
Analytes may also include mono-epitopic liquid species, such as drugs, metabolites, pesticides and pollutants.
Examples of a wide variety of analytes to which the invention is relevant are mentioned in U.S. Pat. Nos. 4,943,522 and 5,624,809, both of which are incorporated herein by reference.
Other analytes include immunoglobulins IgA, IgG, IgE and IgM, enzymes and receptors inclusive of nucleic acids and proteins as described in U.S. Pat. No. 5,624,809.
The term xe2x80x9ctracerxe2x80x9d as made herein may comprise any suitable binding partner specific for analyte(s) of interest contained in a sample being tested. A convenient form of tracer is an antibody specific for an antigen when the analyte of interest. The label may be any conventional marker which after binding of the analyte to the tracer provides a detectable signal. Examples of labels may be a radioactive species, fluorescent species, chemiluminescent species or an enzyme for which a substrate convertible to a coloured product exists such as a peroxidase or alkaline phosphatase. Examples of appropriate tracers or labels are referred to in U.S. Pat. No. 5,208,143, which is herein incorporated by reference, or U.S. Pat. No. 5,624,809 supra.
The preferred label in relation to the present invention is colloidal gold which may be conjugated to antibody or paratope thereof or antigen or epitope thereof. Also may be used as labels are coloured microparticles, coloured dendrimers or coloured dendrimer type molecules.
The term xe2x80x9creceptor ligandxe2x80x9d as used herein refers to any species which may be immobilized or bound to a filter zone or flow zone and which is a specific binding partner for a xe2x80x9ctarget ligandxe2x80x9d. Examples of receptor ligands are antibodies, antigens including haptens, dendrimers, dendrimetric type molecules, oligomers comprising a sequence of monomers selected from the group consisting of amino acids, carbamates, sulfones, sulfoxides, nucleotides, carbohydrates, ureas, phosphonates, liquids, esters or combination thereof or any other molecule or composition capable of recognizing a particular site of a molecule which may be an epitopic or determinant site. Naturally occurring receptor ligands also include nucleic acids, metal chelates, enzymes, biotin, lectins, oligonucleotides and peptides. Dendrimers or dendrimeric-type molecules may also be used as receptor ligands.
The term xe2x80x9ctarget ligandxe2x80x9d as used herein refers specifically to the specific binding partner to the xe2x80x9creceptor ligandxe2x80x9d and thus may include any of the species discussed above for the receptor ligand. It will also be appreciated that the xe2x80x9ctarget ligandxe2x80x9d may also be bound to a specific carrier such as dendrimers, microparticles, latex beads, dendrimers, dendrimeric-type molecules, branched polymers, zeolites, fullerenes, streptavidin, avidin or other substance capable of conjugation to the target ligand.
The microparticles when used as carriers may be of any convenient size that are suitably small enough that they may form a suspension in aqueous solution to facilitate their coating with an antibody, antigen, oligonucleotide or any of the species discussed above for the receptor ligand or target ligand. The average individual size of the microparticles is about 0.01-50 microns, more preferably 1-10 microns. The microparticles can be selected from any suitable type of particulate material, such as polystyrene, polymethylacrylate, polypropylene, silica, teflon, fluropolymers, latex, polytetrafluoroethylene, polyacrylanitrile or polycarbonate.
Reference may also be made to Australian Patent Application Nos. 22431/95 or 22889/95 in the name of Dade International, Inc. or U.S. Pat. No. 5,714,166 for the types of dendrimers that may be used as carriers. Therefore, Australian Patent Application Nos. 22431/95 and 22889/95 and U.S. Pat. No. 5,714,166 are incorporated herein by reference.
The attachment between the receptor ligand and the target ligand may utilize covalent, coulombic, hydrophobic (inclusive of hydrogen binding) or chelation type association between mating functional groups of the target ligand and receptor ligand. An example of such functional groups are thiol groups that bond together to form an xe2x80x94Sxe2x80x94Sxe2x80x94 bond.
A coulombic interaction is an ionic interaction between two oppositely charged species. For example, dendrimeric polymers with a positive or negative overall charge may be attracted to a receptor ligand with an opposite charge.
Chelation type associations make use of metal ions such as, but not limited to, the metals Fe, Co, Ni, Ru, Rh, Pd, Os, Ir, Pt, Pb, Sn, Ge, Sc, Y, lanthanides and actinides, B, Al, Ga, In, Tl, Li, Na, K, Rb, Cs, Fr and Be, Mg, Ca, Sr, Ba, Ra and transition metals. Metal ions immobilized onto solid supports such as membrane filters or microparticles may comprise receptor ligands which will chelate with appropriate coordinating groups on the target ligand. An example of a receptor ligand is iminodiacetic acid complexed with Ni2+ binds with target ligands that have an affinity for Ni2+ acid, such as polyhistidine.
The term xe2x80x9canalyte receptorxe2x80x9d as used herein refers to any specific binding partner for the analyte and thus may comprise antibodies, antigens, oligonucleotides, peptides, proteins or other species discussed above for the receptor ligand or target ligand.
The term xe2x80x9canalyte receptor-target ligand conjugatexe2x80x9d as used herein refers to the analyte receptor when conjugated to the target ligand and thus may comprise, for example, a peptide, nucleic acid or combinations of species of analyte receptor referred to above and target ligand referred to above. This term may also refer to the presence of a carrier as described above in relation to the target ligand.
In another aspect of the invention, there is provided assay apparatus comprising a plurality of discrete flow zones characterized in that there is provided an incubation zone upstream of one or more capture zones which only incorporate immobilized receptor ligand which does not bind directly to analyte in use.
The assay apparatus may comprise a lateral strip as shown hereafter in the illustrated embodiment and thus comprise a plurality of discrete flow zones arranged linearly or in end-to-end, overlapping or abutting relationship.
Preferably the assay apparatus also incorporates a wicking chamber for entrapment of liquids after they have passed through the plurality of discrete zones.
More suitably, however, the assay apparatus may comprise a strip of annular form arranged into each of the discrete zones. Advantageously, there is provided a multiplicity of different capture zones with each capture zone having immobilized thereto a different receptor ligand which does not bind directly with analyte in use. Preferably each of the capture zones is separated from each other by incubation zones.
These also may be provided an incubation chamber or vessel which contains the sample and each of the reagents described above. The mixture of sample and reagents may then flow to an initial zone of the assay apparatus before passing though the or each capture zone before passing into the wicking chamber.
The assay apparatus of the invention comprises, in a preferred form, a plurality of filter discs preferably spaced by spacer discs wherein each of the filter discs are contained in a cartridge. The cartridge may be located above the wicking chamber containing wicking material.
It will also be appreciated that the filter discs or annular or linear strips referred to above may be formed from filter material.
Typical filter materials comprise hydrophilic and liquid permeable polymers inclusive of polyvinylidene fluoride, polyamide, polyester cellulose acetate and, nitrocellulose. Use may also be made of fibres formed from polyvinyl chloride, teflon or other fluoropolymers, polysulfone, nylon, polycarbonate, paper, glass, quartz, silica, ceramic or metallic materials or combinations thereof. Use may also be made of wicking materials referred to hereinafter.
The incubation chamber or vessel discussed above may be in the form of wells which are cylindrical, conical, square or other shape dependent upon the requirements of the user. The wells are suitably formed from a substantially rigid, water insoluble, fluid-impervious, thermoplastic material chemically unreactive with assay reagents and samples. Suitable materials include polyvinyl chloride with or without copolymers, teflon or other fluoropolymers, polyethylene, polystyrene, and the like. The preferred material is polystyrene.
Any convenient bibulous absorbent solid material may be formed as the wicking material which allows for capillary transport of liquids from the filter material. Various materials include cellulose particles, silica gel, cellulosic beads, glass fibres, or filter paper. Preferably the wicking material is porous and can be moulded into complex sizes and shapes from thermoplastic polymers including light density polyethylene, ultra high molecular weight polyethylene, polypropylene, polyvinylidene fluoride, polytetrafluoroethylene, nylon, polyethersulfone and ethyl vinyl acetate.